PROJECT SUMMARY/ABSTRACT Mitochondria are subcellular compartments that are critical for energy production, cell signaling, and the biosynthesis of protein cofactors in higher eukaryotic cells. The mitochondrial DNA (mtDNA) genome is indispensible for mitochondrial function because it encodes protein subunits of the electron transport chain and a full set of transfer and ribosomal RNAs. MtDNA degradation is an essential mechanism in mitochondrial genomic maintenance. In addition, mtDNA degradation is an important quality control measure to cope with mitochondrial DNA damage sourced from endogenous and environmental chemicals. The mechanism of mtDNA degradation and factors involved are poorly understood, which represents a significant knowledge gap. Such knowledge is fundamental to the understanding of mitochondrial genomic maintenance and pathology, because mtDNA degradation may contribute to the etiology of mtDNA depletion syndromes and to the activation of the innate immune system by circulating mtDNA. The objective of this project is to define the chemical and molecular basis of damaged mtDNA degradation and to clarify the role of a major transcription factor and DNA packaging protein TFAM (mitochondrial transcription factor A) in DNA degradation and repair. Addressing this critical knowledge gap will facilitate the PI's long-term goal of unraveling the basis of mitochondrial DNA turnover and its role in mitochondrial pathobiology. This project focuses on a ubiquitous DNA lesion and central DNA repair intermediate, i.e. abasic (AP) sites. The central hypothesis of this application is that TFAM modulates the stability of AP lesions and mediates AP-DNA degradation. This hypothesis is grounded in both strong preliminary data and empirical evidence. Preliminary results will be further evaluated by using a combination of quantitative biochemical, computational, and cellular approaches. Specifically, this research program will delineate the chemical and kinetic basis of TFAM-mediated AP-DNA destabilization, describe the involvement of TFAM in AP-DNA degradation in human cells, and clarify the regulatory role of TFAM in mtDNA repair. The expected outcome is that the project will fill a critical knowledge gap concerning the chemical and molecular mechanisms of mtDNA degradation and novel protein factors involved in the process. This application builds on the PI's strong background in DNA and protein biochemistry, mechanistic enzymology, and quantitative analysis, and accelerates the progress in an exciting, productive area of research into mitochondrial biology. The significance of this project is that it will, for the first time, define the chemical and molecular basis of an mtDNA-degradation pathway and the role of TFAM in mtDNA degradation and repair. Considering that AP sites are key intermediates in mtDNA repair, our insights into AP- DNA degradation will have broad implications for understanding mitochondrial genomic maintenance and instability. New knowledge gained from this research will profoundly advance the field of mtDNA maintenance and potentially inform the development novel therapeutics for mitochondrial diseases.